Date of Award

8-17-2010

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

First Advisor

Sherman Weissman MD

Abstract

INDUCTION OF PLURIPOTENCY IN HUMAN KERATINOCYTES THROUGH mRNA TRANSFECTION. Robert D. Leone, Peter M. Rabinovich, Eugenie Cheng, Efim Golub, and Sherman M. Weissman. Department of Genetics, Yale University, School of Medicine, New Haven, CT. Induced pluripotent stem (iPS) cells are epigenetically reprogrammed somatic cells that exhibit developmental and proliferative characteristics of embryonic stem (ES) cells. Other than alterations made during the reprogramming process, iPS cells are genotypically identical to donor tissue, giving them significant potential in regenerative medicine, basic biology of genetic disease, and drug development. Presently, iPS cell derivation largely relies on the introduction of reprogramming factors (eg, OCT4, SOX2, KLF4, c-MYC) directly into cellular genomes, leaving cells vulnerable to insertional mutagenesis and persistent expression of oncogenic transcription factors. This severely limits their use in clinical and research settings. Here we describe the reprogramming of human keratinocytes through the introduction of exogenous mRNA transcripts. mRNA presence within cells is transient and is unlikely to have permanent effects on the cellular genome, thus avoiding the pitfalls of present methods of iPS cell generation. Several benchmarks have been achieved toward this end, includ-ing: 1) successful expression in human cells of reprogramming factor proteins through the introduction of exogenous mRNA transcripts; 2) phenotypic transformation of human keratinocytes toward ES cell morphology by transfection with mRNA reprogramming factors; and 3) alkaline phosphatase activity (a well described early marker of pluripotency) in a small proportion of transformed cells. These results imply that mRNA transfection may be a viable method for reprogramming somatic cells towards pluripotency.

Share

COinS